Optical, structural and antibacterial properties of phase heterostructured Fe2O3–CuO–CuFe2O4 nanocomposite

The synthesis of the Fe2O3–CuO–CuFe2O4 nanocomposite was effectively achieved through the sol–gel technique, utilizing ethanol as a reactive fuel. Investigation of the nanocomposite’s structure via X-ray Diffraction confirmed the coexistence of Fe2O3, CuO, and CuFe2O4 phases within the material. The Scherrer equation was applied to determine an average crystallite size ranging from 60 to 95 nm. UV–visible spectroscopy studies suggested the material possesses an approximate energy bandgap of 4 eV. Scanning Electron Microscopy provided insights into the nanocomposite’s surface morphology, which exhibited a porous and heterogeneous aggregation of particles in various sizes and shapes. When tested for antibacterial efficacy, the nanocomposite exhibited activity against gram-positive S. aureus with a maximum zone of inhibition (ZOI) measuring 9 mm at the highest concentration, whereas no inhibitory effect was detected against gram-negative E.coli.


Preparation of the sample
The Fe 2 O 3 -CuO-CuFe 2 O 4 nanocomposite (NC) was synthesized using the sol-gel method 20,[35][36][37][38] .Initially, two separate solutions were prepared: one with Cu(NO 3 ) 2 •3H 2 O (5.798 g in 30 mL of ethanol) and another with Fe(NO 3 ) 3 •9H 2 O (9.69 g in 30 mL of ethanol), keeping a molar ratio of 1:1.Each solution was stirred for fifteen minutes at a temperature of 22 ± 2 °C until homogeneity was achieved.These solutions were then combined and stirred continuously at 80 °C for seventy minutes.During this time, the mixture gradually dried while being heated and mixed, eventually forming a gel that turned into a dry mass.Subsequently, this dry mass was ground into a fine powder and annealed at a temperature of 800 °C for 120 min.After completing these procedures, the composite was ready for subsequent characterization.

Antibacterial test
The antibacterial effectiveness of Fe 2 O 3 -CuO-CuFe 2 O 4 NC was evaluated in vitro using the widely recognized disk diffusion method [39][40][41] .The assessment involved testing against specific Gram-positive (S. aureus) and Gramnegative (E.coli) bacterial strains, with azithromycin (AzM) serving as the reference standard 35 .Initially, the bacterial strains were cultured at 37 ± 1°C for 24 h and then adjusted with nutrient broth to achieve an absorbance of 0.075-0.1.Test plates were prepared by spreading the bacteria over MHA media in Petri dishes.Subsequently, 6 mm diameter disks, each wetted with 20μL on both sides containing various concentrations (50, 100, and 200 mg/mL) of Fe 2 O 3 -CuO-CuFe 2 O 4 NC, were placed on the media surface and incubated at 37 ± 1 °C for 20-23 h.The antibacterial activity was assessed by measuring the diameter of the inhibition zone (ZOI) using a ruler and comparing the results with the controls 5 .The experiment was conducted in duplicate, and the average values were reported.

Characterization techniques
The structural properties were analyzed using X-Ray Diffraction (XRD) with a XD-2 X-ray diffractometer employing Cu Kα radiation (λ = 1.54Å), while the morphological properties were examined using SEM (Jeol Ltd., Tokyo, Japan).The optical properties were studied through UV-Vis spectroscopy using a Hitachi U3900 instrument with Varian Cary 50 software.

X-ray diffraction
The X-ray diffraction (XRD) pattern shown in Fig. 1 indicates the formation of Fe 2 O 3 -CuO-CuFe 2 O 4 nanocomposite after being prepared and annealed at 800 °C.The diffraction peaks confirm the presence of cubic Fe 2 O 3 , monoclinic CuO, and tetragonal CuFe 2 O 4 structures, with no impurities or secondary phases present.The specific diffraction peaks and reflection planes for Fe 2 O 3 , CuO, and CuFe 2 O 4 are consistent with JCPDS card No. 39-1346, 45-0937, and 34-0425, respectively 6,9,19,42 .The average crystallite size(D ) of the Fe 2 O 3 -CuO-CuFe 2 O 4 nanocomposite was determined using Scherer's formula (Eq. 1) 43,44 , with the calculated size recorded in Table 1. (1) where λ is the XRD wavelength (1.5406 A), θ is the Bragg diffraction peak (in radian), and β is the full width at half maximum (FWHM).Additionally, the dislocation density (δ) was computed using Eq. ( 2), providing information about vacancies and defects in the crystal.Also, the lattice strain ( ε ) of nanocomposite ε = βcosθ/4 was computed.The variation in the strain is may be due to the alteration in structure, crystallite size and morphology.The estimated dislocation density and the strain values are also recorded in Table 2 35,45,46.Lattice strain often arises due to discrepancies between the ideal and the actual distances of atoms within the crystal lattice, which can result from various synthesis conditions such as temperature, pressure, and the rate of formation.It is a critical parameter that provides insight into the defect density and crystallite size of the material.The lattice strain values suggest the presence of intrinsic stress within the nanocomposite, possibly induced by the heterogeneity of the constituent phases and their interfacial interactions.This strain can be beneficial or detrimental to the material's properties, depending on the specific application.For instance, in certain cases, a higher lattice strain can lead to improved electrical or ionic conductivity due to the creation of more active sites, which is advantageous for catalytic applications or in energy storage devices like batteries and supercapacitors.Conversely, excessive lattice strain might compromise the structural integrity and decrease the mechanical stability of the material.Therefore, understanding the extent and the nature of lattice strain is essential for tailoring the synthesis process to enhance the desired properties of the nanocomposite for specific applications.
The lattice constants (a,b & c), unit cell volume (v), atom number per unit cell(Z), and the mass density(ρ) for the cubic Fe 2 O 3 , monoclinic CuO, and tetragonal CuFe 2 O 4 structures were computed and recorded in Table 2 46,47 .

SEM
The Scanning Electron Microscopy (SEM) analysis was performed to observe and understand the morphology of the prepared sample.www.nature.com/scientificreports/

UV-visible
UV-Visible spectroscopy was utilized to investigate the optical properties of the Fe 2 O 3 -CuO-CuFe 2 O 4 nanocomposite.In Fig. 3, the absorption spectra of the nanocomposite cover the range of 200-1000 nm, indicating that absorption was most prominent at shorter wavelengths and decreased gradually as the wavelength increased.Moreover, in Fig. 3, the UV-Vis absorbance spectrum of the synthesized CuO-Fe 2 O 3 -CuFe 2 O 4 nanocomposite does not exhibit distinct strong absorption peaks, which is characteristic of the nature of the materials studied.
The spectrum shows a significant absorption edge at approximately 290 nm, indicative of the bandgap of the composite.The absence of additional strong absorption peaks can be explained by the following: (i) Homogeneity of the Composite: The sol-gel method used for synthesis provides a uniform material with a consistent phase composition, which leads to broad absorption rather than sharp, localized peaks.(ii)Phase Interactions: The presence of multiple oxides within the composite can lead to interaction between the phases, which may result in a broadening of the absorption features, smoothing out any peaks that could be associated with individual  (v) Surface States: The sol-gel synthesis and subsequent calcination at 800 °C may create a nanocomposite with fewer surface states that could give rise to distinct absorption peaks.Instead, a continuous distribution of states at the band edge results in a smooth absorption spectrum.The absorbance continuously decreases beyond the absorption edge, from 290 nm onwards up to 1000 nm, without showing any additional peaks, which is in agreement with the absorption behavior expected for nanocomposites with the aforementioned characteristics.The spectral profile obtained is consistent with optical transitions and bandgap absorption, and the data supports the composition and structure of the prepared nanocomposite as verified by XRD analysis.Additionally, Fig. 4 illustrates the changes in the absorption coefficient (α) and the extinction coefficient (k) of Fe 2 O 3 -CuO-CuFe 2 O 4 with respect to wavelength.The data shows that the absorption coefficient (α) increases with the wavelength, while the extinction coefficient(k) is highest at shorter wavelengths and decreases as the wavelength increases.Tauc's relation 48,49 was employed to calculate the optical energy band gap, resulting in a value of 4 eV for the Fe 2 O 3 -CuO-CuFe 2 O 4 nanocomposite, as shown in Fig. 5.The energy bandgap of approximately 4 eV in the synthesized CuO-Fe 2 O 3 -CuFe 2 O 4 nanocomposite presents significant implications for its use across various applications.This value, indicative of the material's ability to absorb UV photons, positions the nanocomposite as a promising candidate for photocatalytic activities which could include the degradation of organic pollutants and the production of hydrogen through water splitting.Additionally, the bandgap situated in the visible to UV spectrum enhances the material's suitability for electronic and optical devices, such as UV photodetectors and the active layers of light-emitting diodes that operate within the blue/UV range.The semiconductor properties inherent to a bandgap of this magnitude suggest potential usage in high-power and high-frequency electronic devices, benefiting from the material's good insulation characteristics.Furthermore, a larger bandgap typically confers greater chemical and thermal stability to the material, making it ideal for deployment in environments that may subject devices to extreme conditions.Therefore, optimizing the synthesis process to fine-tune the bandgap is crucial, especially as the interactions among the CuO, Fe 2 O 3 , and CuFe 2 O 4 phases within the nanocomposite contribute to its distinctive electronic properties and the resulting bandgap observed 25,50 .The energy gap value calculated for this nanocomposite aligns with the results obtained by Kaveh Parvanak Boroujeni et al. www.nature.com/scientificreports/from their study on the Fe 2 O 3 /CuO nanocomposite, which demonstrated an energy gap of 4.2 eV 50 .The observed 4 eV energy band gap for the Fe 2 O 3 -CuO-CuFe 2 O 4 nanocomposite, determined using the sol-gel method, likely arises from the intricate interactions and interfaces between its constituent elements.When these materials are combined in a nanocomposite, potential synergistic effects may result from chemical reactions and interfacial interactions among the components.These interactions can lead to modifications in the energy band gap of the nanocomposite, which may not simply be a summation of the band gaps of the individual components due to the emergence of new energy states and the influence of interfacial interactions and structural changes at the nanoscale.

Antibacterial activity
The Fe 2 O 3 -CuO-CuFe 2 O 4 nanocomposite was synthesized to assess its antibacterial efficacy against Staphylococcus aureus (gram-positive) and Escherichia coli (gram-negative).The results, as depicted in Fig. 6 and summarized in Table 3, revealed selective antibacterial activity.The nanocomposite effectively inhibited the growth of S. aureus, as indicated by a zone of inhibition (ZOI) diameter greater than 6 mm, which is considered to be a marker of potent antibacterial action.However, it failed to impede the growth of E. coli, where the ZOI diameter was 6 mm or less, denoting inadequate antibacterial activity.The selective action can be attributed to the structural differences between gram-positive and gram-negative bacteria.Gram-negative bacteria possess a less accessible, negatively charged peptidoglycan layer and a lipopolysaccharide-rich outer membrane that acts as a barrier to the nanocomposite's active agents.Conversely, gram-positive bacteria are more vulnerable due to their thicker peptidoglycan layer which lacks such a protective barrier, enabling the nanocomposite's active components to penetrate and exert their antibacterial effects 40,43,51 .The antibacterial properties of the Fe 2 O 3 -CuO-CuFe 2 O 4 nanocomposite are primarily due to two mechanisms: the generation of reactive oxygen species (ROS) and the release of heavy metal ions.The Fenton reaction, facilitated by the nanocomposite under light irradiation, generates ROS, which induces oxidative stress leading to cellular damage.This stress results in intracellular disruptions, manifesting as DNA damage, lipid peroxidation, and protein oxidation, ultimately causing bacterial cell death 44,[52][53][54] .Furthermore, the positively charged Cu 2+ and Fe 3+ ions from the nanocomposite interact electrostatically with the negatively charged bacterial cell wall, resulting in protein denaturation and disruption of DNA replication processes.These combined effects culminate in the inhibition of bacterial growth and cell death, elucidating the antibacterial mechanism of the Fe 2 O 3 -CuO-CuFe 2 O 4 nanocomposite without adversely affecting nonbacterial cells [55][56][57][58][59][60] .A comparison of inhibition zone of the fabricated CuO-Fe 2 O 3 -CuFe 2 O 4 nanocomposite with other multi-metal oxides from literature is given inTable4.To facilitate comparison, the test concentration and the method used were also tabulated.Figure 7 shows the mechanisms of the antibacterial activity of the nanocomposite.In the discussion of the results presented in Table 4, it is important to contextualize the observed  Zone of Inhibition (ZOI) values within the framework of our specific experimental parameters.While it is acknowledged that the ZOI values observed in our study may be lower than those reported in other literature, it is imperative to consider the unique conditions under which our experiments were conducted.Factors such as the concentration of the nanocomposite, the incubation times, the specific bacterial strains used, and the methodology employed are all variables that can significantly impact the ZOI measurements.Moreover, it should not be automatically inferred that lower ZOI values equate to reduced antibacterial effectiveness.The diffusion rate of the nanocomposite within the agar, the interaction dynamics between the nanocomposite particles and the bacterial cell walls, as well as the potential for synergistic or antagonistic effects arising within the media, are all critical factors that need to be taken into account.These elements can considerably influence the antibacterial activity outcomes and may provide an explanation for the ZOI values obtained in our experiments.As such, the efficacy of the antibacterial properties of a substance should not be assessed solely based on ZOI values but should also consider the complex interplay of the aforementioned factors.The interplay between the optical characteristics of our Fe 2 O 3 -CuO-CuFe 2 O 4 nanocomposite, namely the absorption edge at approximately 290 nm correlating to a bandgap of 4 eV, and its selective antibacterial efficacy against gram-positive bacteria, is a focal point of our study.This specific optical threshold implicates the nanocomposite's proficiency in harnessing UV radiation to generate reactive oxygen species (ROS), potent agents of oxidation.These ROS are instrumental in attacking bacterial cell structures, particularly the less-protected  We propose that the unique composition and structure of the nanocomposite promote a photocatalytic effect under UV illumination, which leads to the generation of ROS.These oxidative species are theorized to permeate the cell wall of gram-positive bacteria more readily than the more robust outer membrane of gram-negative bacteria, such as E. coli.

Conclusion
The synthesis of Fe 2 O 3 -CuO-CuFe 2 O 4 nanocomposite (NC) via sol-gel method was discussed.Ethanol was utilized as a capping agent to prepare NC.XRD, SEM, and UV-VIS Spectroscopy were employed to characterize the properties of the prepared material.XRD studies revealed that Fe 2 O 3 , CuO and CuFe 2 O 4 exhibit cubic, monoclinic, and tetragonal phases respectively.UV-visible studies of the nanocomposite showed an energy bandgap of about 4eV.The nanocomposite exhibited antibacterial activity against gram-positive bacteria only.
Based on the aforementioned evidence, Fe 2 O 3 -CuO-CuFe 2 O 4 nanocomposite can be a suitable material for optoelectronic devices, diagnostic and therapeutic applications.

Figure 2
displays the SEM images of the synthesized nanocomposite at different magnifications.Analysis of Fig. 2 confirms the presence of Fe 2 O 3 -CuO-CuFe 2 O 4 nanocomposite, revealing numerous porous agglomerates with irregular sizes and shapes.

Table 4 .
A comparison of antibacterial analysis of CuO-Fe 2 O 3 -CuFe 2 O 4 NC with some other nanocomposites.www.nature.com/scientificreports/peptidoglycan layers of gram-positive species such as S. aureus, resulting in notable cell damage and inhibition.